The virus family filoviridae consists of two recognized viruses, Marburg virus (MBGV) and Ebola Virus (EBOV). Filoviruses are among the deadliest of acute virus infections in humans and nonhuman primates and are sufficiently contagious to cause sequential infections of persons exposed to blood and body fluids. MBGV causes severe hemorrhagic fever characterized by high mortality rates in both human and nonhuman primates. The first recognized infection of humans by a filovirus occurred in 1967 when simultaneous outbreaks of hemorrhagic fever occurred in Marburg and Frankfurt, Germany, and Belgrade, Yugoslavia. All cases were associated with laboratory workers engaged in processing kidneys from African green monkeys for cell culture production. There were 31 recognized infections and 7 deaths. Two cases of hemorrhagic fever caused by a virus similar to the 1967 MBGV were identified in Kenya in 1980. In 1987 MBGV was isolated from a fatal hemorrhagic fever case, and was subsequently shown to differ substantially from the original MBGV isolate.
Human outbreaks, though relatively few, have involved scores and sometimes hundreds of people, the viruses often infecting health care professionals and wreaking havoc on local medical infrastructures. Yet, in the context of global health statistics, filoviral disease incidence has been insufficient in the past to prompt aggressive vaccine development. After the 9/11 terrorist attack, widespread vaccination is logically assumed in case of its use as a bioterrorism agent. Moreover, these viruses have additional traits often associated historically with biological weapons, such as high infectivity in aerosol form, stability in aerosol, and even reports of active weaponization. Searches for knowledge of the scientific requirements for vaccine development were reduced to a combination of curiosity and insurance against unknowns, e.g., the possibility of viral mutation to fully contagious or even pandemic spread; or the possibility of viral emergence in new geographical areas or host species outside the currently unknown ecological niches in sub-Saharan Africa.
Several isolates of MBGV have been adapted to uniform lethality in strain 13 guinea pigs. MBGV (Musoke), MBGV (Ravn), and MBGV (Ci67) were adapted to adult strain 13 guinea pigs via serial passages of a non-guinea pig adapted virus through guinea pigs. All guinea pig adapted MBGV produce uniform lethality in both strain 13 and Hartley guinea pigs. The glycoprotein is the only membrane protein exposed on the viral surface of Marburg virus, strain Musoke, and has been isolated by expression in E. coli and identified by its immunoreactivity with specific antisera. GP thus appeared to be an attractive target for neutralizing antibodies. Antigenicity, and vaccine potential of MBGV GP expressed by baculovirus recombinants, was also examined. This recombinant truncated GP elicited protection against lethal challenge with the MBGV isolate from which it was constructed and less effectively against an antigenically disparate MBGV isolate; killed (irradiated) MBGV antigen was protective, in a reciprocal fashion, against MBGV types.
Previous vaccine studies have demonstrated that whole, irradiated MBGV, when used to vaccinate guinea pigs in the presence of the RIBI adjuvant, results in complete protection from disease and death. In turn, the glycoprotein (GP) antigen has emerged as an important and perhaps necessary component of efficacious vaccines. Both antibodies and T cells appear to have roles in filovirus immunity, but significant gaps remain in the understanding and measurement of vaccine-induced immune responses that prevent or mitigate filovirus disease.
Immune stimulants play an important and essential role in development of vaccine adjuvants and drug delivery systems. Subunit vaccines are designed to include only the antigens required for protective immunization and to be safer than whole-inactivated or live-attenuated vaccines. However, the purity of the subunit antigens and absence of immunomodulatory components often result in weaker immunogenicity.
Adjuvants are a heterogeneous group of compounds that are capable of triggering or enhancing an immune response against an otherwise poorly immunogenic entity and can be broadly separated into two classes based on their principal mechanisms of action; (a) vaccine delivery system and (b) immunostimulatory adjuvants. Vaccine delivery systems are generally particulate (e.g., emulsions, microparticles, ISCOMs and liposomes), and mainly function to improve the uptake of antigens by the immune system, and stimulate antigen-presenting cells (APC) to assist in mounting an immune response. Immunostimulatory adjuvants are predominantly derived from pathogens and often pathogen associated molecular patterns (PAMP), which activate cells of the innate system and then focus the acquired immune response. In some studies, delivery systems and immunostimulating agents have been combined.
There are several features that are necessary for an adjuvant to be effective: (a) it should be non-toxic or at least have a wide therapeutic range (effective dose<<toxic dose); (b) it should form a depot at the injection site, (c) it should increase antigen (or antigen-adjuvant complex) uptake into APCs; (d) it should mobilize APCs so that they travel to the draining lymph nodes; (e) it should up-regulate the necessary additional signals on APCs to prime T-cells (CD4+ and CD8+) efficiently, (f) ideally, different adjuvants would lead to either a preferential humoral (antibody, Th2-like) or cell mediated (CTL, Th1-like) immune response. Several synthetic and other defined adjuvants are used or are currently being tested and evaluated. These include; alum, poloxamers, MF59, lipopeptides, the synthetic peptide analog PAM3Cys, muramyl dipeptides and derivatives, CpG oligonucleotides, polycationic peptides, carbohydrate polymer derivatives of mannan, chitosan, 1,3-β-glucans, etc. and saponins.
Alum is not capable of generating cytolytic T-lymphocytes (CTL) that are prerequisite for the clearance of intracellular pathogens. This inability to generate CTL is due to the manner in which the immune system responds but produces >90% Th2 response.
Saponin-based adjuvants such as Quil-A, QS-21 and GPI-0100 have been applied to a variety of vaccine formulations because of the ability to promote CTL and Th1 responses. Saponins are a heterogeneous group of sterol glycosides or triterpenoid glycosides that are present in a wide range of plant species. Specially, triterpene saponins have been identified with strong immunoenhancing capacity and toxicity, but they cause strong local reactions at the site of injection mainly due to their lytic properties. In particular, the saponins from Quillaja saponaria Molina, Gypsophila sp. and Saponaria officinalis have unique immunostimulating and immunomodulating properties. An enriched heterogeneous mixture of saponins named Quil-A was extracted from the Quillaja bark that possessed adjuvant properties. The toxicity profile of Quil-A precludes expanded use in human vaccines but is being used commercially in a veterinary vaccine. The four most predominant saponins (QS-7, QS-17, QS-18 & QS-21) and about 23 other minor saponin contents from Quillaja saponaria extracts were purified to near homogeneity by reversed-phase HPLC.
The characteristic feature of these saponins is the presence of an aldehyde group at C-4 of the aglycones (quillaic acid and gypsogenic acid). Generally, these saponins are bidesmosides with branched sugar substitution at C-3 and C-28. The presence of a 3,5-dihydroxy-6 methyloctanoic acid ester on C-28 fucose residue is unique to Quillaja saponins and plays a critical role in producing cytotoxic T-lymphocyte (CTL) responses. Among all Quillaja saponins, single isolated and purified QS-21 has been shown to have the most potent adjuvant activity, as a complex amphipathic molecule that readily forms micelles. QS-21 is potent for CTL induction, inducing Th1 cytokines (IL-2, IFN-γ) and antibodies of the IgG2a isotype and is undergoing several clinical trials as an adjuvant for cancer vaccines (melanoma, breast and prostate), and infectious diseases (HIV-1, influenza, herpes, malaria, hepatitis B). Doses of 200 μg or higher of QS-21 in humans have been associated with significant local reactions, but lower doses appear to be well tolerated. The other main drawback of QS-21 is pH dependent stability. Due to acylated octanoic side chain, the stability is a problem leading to deacylation resulting in loss of overall adjuvant activity and degradation on storage at ambient temperature.
The purification and characterization of adjuvant active saponins from Quillaja saponaria has also enabled a correlation between structure and function. The removal of the fatty acid domain to deacylated saponins resulted in the loss of production of antibody and CTL-stimulating capacity. Similarly, reduction of the C-4 aldehyde or blocking it with amine caused this same loss of activity. This clearly suggests that this functional group is critical for the adjuvant's activity, probably by interacting with free amino groups on the surface on a lymphocyte or APC. Also, the aldehyde may stabilize a saponin-antigen complex through imine formation. All the saponins contain a single glucuronic acid that may impart an overall anionic charge to the saponins at physiological pH, as the conversion of acid groups to amides does not affect overall adjuvant activity. In general, for strong adjuvant activity of oleanolic acid-containing saponins, three factors are critical to stimulate antibody and CTL production: a) presence of an aldehyde group, b) presence of a fatty acid chain, and c) sugars (for receptor activity and micelle formation).